Fluorescent Gold Nanoparticles for Cellular Biomedical Applications

Stetson University Department of Chemistry Fluorescent Gold Nanoparticles for Cellular Biomedical Applications Jaime Fields Independent Research Deborah B. Maxwell Ph. D. April 11, 2016 2 Abstract: Colloidal gold nanoparticles demonstrate promising potential towards applications in cellular imaging due to their biocompatibility, low-toxicity, and optical properties. Other metallic nanoparticles have been extensively studied because of their ideal optical properties, such as a wide excitation and narrow emission spectra, and ease of biological interactions due to small particle size.3 Gold nanoparticles offer an encouraging alternative to other, often toxic, metallic nanoparticles due to their low toxicity and history of medicinal use. I synthesized gold nanoparticles using a traditional Turkevich method of a citrate reduction of a gold precursor. The synthesized gold nanoparticles were characterized, with size and fluorescence being of key importance. By interacting the fluorescent gold nanoparticles with BSA there is potential for their development as a dual imaging agent, drug delivery vehicle. Introduction: Records of the use of soluble gold for medicinal purposes can be found dating back to the 17th century; information on the use of colloidal gold solutions for the treatment of various diseases is found in the writings of Dr. Fracisci Antonii published in 1618.1 Some suggest that medicinal use of colloidal gold dates back even further, to the Chinese and Egyptians around 2500 BC.2 In the last decade, with the advancement of medical imaging, gold nanoparticles may have found a potential new use as a fluorescent probe. Metal nanoparticles of various forms have already been extensively researched with emphasis on biomedical applications due to their small size allowing for interaction with biomolecules. Gold nanoparticles have been thought to provide advantages over their metal-based counterparts due to their low toxicity, which is demonstrated by the history of medicinal gold usage. Furthermore, it has been shown that functionalizing the 3 surface of such nanoparticles could allow for selective interaction with cell membranes, cancer cells, and more.3 For example, in a study conducted by Chen et al., gold nanocages that were bioconjugated with antibodies successfully targeted breast cancer cells for early-stage tumor diagnostics.4 The physical properties of nanoparticles such as size, concentration, and ligand functionalization determine fluorescence ability as well as the extent and form of biological interactions. Fluorescent semi-conductor nanoparticles, also known as quantum dots (QDs), are shown to have optical advantages over traditional contrast agents due to their broad absorption spectra, meaning their ability to be excited by a wide range of wavelengths, and their narrow emission spectra. These optical characteristics combined make them ideal for multi-plexed imaging, which means that various QDs could be introduced to a cellular environment and QDs of different emission wavelengths could be activated with the same wavelength of light producing a multicolored image. QDs also have the advantage of long fluorescent lifetime and stability, which enables them to undergo multiple excitation cycles, allowing for images to be obtained several times within the lifetime of the nanoparticles.5 Figure 1: Diagram comparing the excitation (a) and emission (b) spectra of Cadmium quantum dots and the organic dye rhodamine. The diagram shows the wide excitation spectrum of the QDs and the narrow emission spectrum [6]. 4 The fluorescence property of QDs is a primary characteristic that encourages nanomedical research; it has been found that QDs may be significantly brighter than organic dyes.7 Fluorescence is mainly determined by nanoparticle size and shape, which can be controlled through experimental conditions during synthesis, such as temperature, reaction time, reagents, and sometimes capping agents. A stability factor of nanoparticle solutions that is extremely important to biological applications is a minimization of aggregation, or clustering of particles. It is important to minimize aggregation of the nanoparticles as aggregation in a biological system could result in both a loss of nanoparticle function and capillary blockage.2 Aggregation free, citric acid stabilized spherical gold nanoparticles diameters 14, 30, 50, 74, and 100 nm were incubated for 6h with HeLa cells by Chithrani et al. (2006). Maximum cell uptake into the cell cytoplasm via receptor-mediated endocytosis occurred with the 50 nm particles and the adsorption of proteins to the nanoparticle surface improved uptake.8 Their experiment shows that citric acid stabilized nanoparticles may have a promising future in the realm of cellular imaging and targeted drug delivery. In regards to targeted drug delivery, the use of gold nanoparticles as drug carrier systems is a promising area of research. Gold nanoparticles are said to be an ideal carrier system due to their ease of synthesis and non-toxic core, as discussed in regards to their use as a fluorescent probe as well. Gold nanoparticles have the ability to carry a large variety of conjugates, ranging from small drug molecules to large biomolecules such as proteins, which is typically achieved through surface modification.9,10 Two key approaches to drug delivery are active and passive targeting. Passive targeting is the collection of the drug carrier system in a specific location due to factors such as size, surface charge, or natural biological pathways. Active targeting on the other hand employs modification of the drug carrier system to target and bind to specific cells or 5 tissues, such as the adsorption of the protein transferrin onto the gold nanoparticle surface achieved by Chithrani et al. (2006).11 Because citric acid stabilizers are weakly bound to the nanoparticle surface, they can be replaced with proteins, allowing for cellular uptake in the form of receptor mediated endocytosis.8 Both active and passive drug delivery targeting using gold nanoparticle carrier systems are likely areas of future biomedical research. In our study we synthesized fluorescent gold nanoparticles with the goal of protein conjugation for potential application as a drug carrier system with cellular imaging capabilities. Experimental Methods: Gold nanoparticle synthesis was completed according to the procedure of Turkevich et al., a reduction of a gold precursor with trisodium citrate.12 In initial trials we used the reaction time and temperature of the Turkevich synthesis conducted by Polte et al. (2010). Gold nanoparticles were synthesized from the addition of a 35 mL solution of gold chloride (7 mg HAuCl4 x 3H2O, Aldrich) and a 35mL solution of trisodium citrate (51.45 mg Na3C6H5O7 x H2O, Aldrich) that were preheated to the reaction temperature of 85 °C. Reactants were obtained from Sigma Aldrich. The citrate solution was added to the gold chloride while under stirring in a 3 neck round bottom flask in a temperature controlled water bath fitted with a thermometer, condenser, and addition funnel. The synthesis was allowed to react for 42 minutes followed by a quenching procedure; the nanoparticle solution was stored for analysis.13 6 Figure 2: Synthesis setup. The left image is the trisodium citrate solution warming to reaction temperature. The image on the right shows the reaction vessel containing gold precursor in a temperature controlled water bath at 85 °C. Preliminary synthesis: We conducted a preliminary trial using the synthesis method as described; the resulting solution was a colloid of deep purple color that showed no signs of aggregation several weeks after formation and very slight aggregation 5 weeks after formation, indicating stability of the particles in solution. Characterization of the particles resulting from the preliminary trial will be addressed later in this paper. Experimental Methods Continued: After several trials using the aforementioned method of synthesis without yield of fluorescent AuNPs, a new approach was taken with the aim of synthesizing smaller AuNPs. AuNPs were synthesized using a simplified Turkevich method in which .00788 g of gold chloride was dissolved in 20 ml of distilled water and brought to a boil on a bench-top hotplate in a 50 ml Erlenmeyer flask under continuous stirring. 0.5 g of trisodium citrate was dissolved in 50 ml of 7 distilled water, of which 2 ml was added to the boiling gold chloride solution. The reaction was allowed to run at high heat for several minutes (various trials conducted: 8min or 4min), and then quenched over ice until returned to room temperature. Data Collection: UV-Vis Spectroscopy: Nanoparticles were characterized after synthesis to determine size, concentration, and fluorescence. Using tabular data obtained from Haiss et al. (2007), the size and concentration of our gold nanoparticles were determined directly from a UV-Vis spectrum. Tables give nanoparticle diameter in nanometers for uncoated spherical gold nanoparticles as a function of the absorbance. Our preliminary synthesis showed an absorbance peak at a wavelength of 526.00 nm. From the tabular material, that wavelength of the absorbance of the surface plasma resonance peak (Aspr) corresponds to particles of 36 nm in diameter. Additionally, the molar extinction coefficient (ε) at 450 nm was determined from tabular data; it is presented as a function of particle diameter. The concentration was calculated using the Beer-Lambert law where c is concentration in molarity and l is the path length, which is 1.00 cm in our experiment.14 Beer-Lambert Law: A= (ε)(c)(l) (equation 1) Atomic Force Microscopy: The UV-Vis spectroscopy results and calculations were used to give us an idea of the size of the nanoparticles before we image them with using atomic force microscopy (AFM). AFM can 8 provide scans of the particles on the nanometer scale down to the tens of nanometers range with great accuracy. AFM scans the sample surface using a probe tip with a radius of curvature in the nanometer range that is attached to the end of a cantilever. The probe tip interacts with the sample at very close proximity, and therefore experiences either repulsive or attractive forces that result in a bending moment of the probe, which then causes the cantilever to experience a deflection that is measured using a laser beam. Figure 3: Diagram of the AFM. The cantilever tip is interacting with the surface of the sample, resulting in deflections of the cantilever. Interactions are measured using a laser that hits the back of the cantilever and is reflected onto a photodiode detector [16]. For the imaging of nanoparticles, intermittent contact mode should be used. In this scan mode, the probe is brought very close to the sample and the cantilever is oscillated in a manner that allows for atomic forces between the probe and the surface to be measured, allowing surface height to be determined. The resulting images are a topographic representation of the sample surface. While the lateral area of the particle may be subject to distortion, the particle height, z-axis, is measured to a very accurate degree.15 9 Sample preparation: There are several methods of sample preparation that are suitable for the scanning of nanoparticles with the AFM instrument. In order to be measured with the AFM, the nanoparticles must be dispersed onto a flat surface, such as a clean glass slide or, preferably, atomically flat mica. The nanoparticles were dispersed using a method called spin coating, in which a drop of sample is place onto the slide, then rotated at a high rate in order to disperse the sample across the slide.16 AFM of our gold nanoparticles from the preliminary trial: After determining that our nanoparticles were of suitable size to be observed with the AFM instrument, we prepared slides to be scanned. Drops of gold nanoparticle solution were placed onto three slides: glass, glass with double stick tape, and mica. Each slide was then rotated for 30 seconds at a rate of 634 rotations per minute. The slides were covered to protect from dust and debris for the following two weeks while scans were obtained. Scans showed that our nanoparticles were in the 300 nm diameter range in the x-y plane, and roughly 1/3 of that size along the z-axis. Although AFM measurements are thought to be most accurate along the z-axis, there is still a significant size discrepancy from our expected nanoparticle size based on synthesis methods and UV-Vis characterization. 10 Figure 4: These scans show a topographical view of the surface of the glass slide with a spin-coated layer of our synthesized nanoparticles. The image to the left shows a cross-sectional area of 10.0 μm x 10.0 μm and the image to the right shows a cross-sectional area of 2000 nm x 2000 nm focused on a particle 78.50 nm in height. Fluorimetry: A fluorimeter was used to determine the fluorescence of the synthesized gold nanoparticles. Particles were excited at 410 nm with an emission range of 350 to 650 nm. The nanoparticles from the preliminary synthesis did not show any fluorescent properties; this however could be a result of the large size of the particles. Later syntheses using the simplified Turkevich method resulted in the scans shown below. Results: Figure 1: Uv/Vis spectrum of AuNP Solution, maximum at 520.50 nm. Figure 2: Fluorescence spectrum of AuNP solution (4 minute synthesis) at a 1 to 3 dilution with 410 nm excitation. 11 Figure 3: AuNP solution following 4 minute reaction time. Figure 4: AFM image of dispersed AuNP solution on a mica slide. Measurements demonstrated a particle diameter of up to 32.05 nm. Figure 6: UV/Vis Spectra of pure AuNP solution and BSA/AuNP mixture, maxima correspond to 520.50 nm and 524.50 nm respectively. Figure 5: AFM image of dispersed AuNP solution on mica, particle diameter to 20.83 nm. Figure 6: Fluorescence emission scan of pure AuNP solution, 410 nm excitation. Figure 7: Fluorescence emission scan at 280 nm excitation of BSA solution, pure AuNP solution, and 1:2 BSA to AuNP mixture. Figure 8: Fluorescence emission scan at 410 nm excitation of BSA/AuNP mixture with a large excess of BSA. 12 For the 4-minute synthesis time, the UV/Vis maximum corresponded to a particle diameter and concentration of 19 nm and 2.034E-9 M respectively. AFM scans were consistent with this particle size, showing a particle diameter of 20.83 based on the z-axis measurement in figure 5. Fluorescence resulted from excitation at 410 nm. The addition of BSA resulted in a shift of the UV/Vis spectrum, indicating particle size change. Additionally, emission scans showed alteration of AuNP fluorescence upon BSA addition, however results are inconclusive. Discussion: With the simplified synthesis method and shorter reaction time, fluorescence of the AuNPs was achieved. The synthesized particles were of desired size and fluorescence for cellular applications. The particles were conjugated to BSA in an effort to examine biocompatibility. The BSA conjugation represents potential application as a fluorescent carrier system for biomolecules. Although nanoparticle size difference upon BSA addition was observed, indicating conjugation, further research is needed to study the effect of this conjugation on the nanoparticle fluorescence. Future work will involve the introduction of conjugated AuNPs to cells with a focus on cellular uptake and cellular imaging. A future goal of this project is to demonstrate cellular uptake of the synthesized AuNPS, particularly with cancer cells. According to He et al., gold nanoparticles prepared using a citrate reduction of chloroauric acid of diameter 16 to 55 nm showed significant luminescent properties and were successfully introduced to cervical cancer HeLa cells through the endocytic pathway, resulting in the imaging of the live HeLa cells. The results of their study indicate that gold nanoparticles have adequate luminescence to be detected by fluorescence instruments and the ability to enter cells through natural biologic pathways. The study also mentions great potential of their gold nanoparticles 13 towards cell imaging due to biocompatibility, photostability, and ease of conjugation chemistry, all of which are promising characteristics for our research goals.16 As a future project, it could be possible to design an experiment synthesizing conjugated gold nanoparticles that will then undergo endocytosis with selected cancer cells, demonstrating a valuable potential biomedical use as a dual imaging agent, targeted drug delivery vehicle. Acknowledgements Thank you to the Stetson University Department of Chemistry for use of instruments and project funding. Thank you to Dr. Deborah Maxwell for her guidance and support throughout this project. I would also like to acknowledge Dr. Kevin Riggs and the Physics Department for usage of the Atomic Force Microscope and Dr. Price for project discussion. 14 References 1 Daniel, M.; Astruc, D. Chemical Reviews, 2004, 104 (1), 293-346. 2 Thakor, A.; Jokerst, J.; Zavaleta, C.; Massoud, T.; Gambhir, S. Nano Lett. 2011, 11, 4029-4036. 3 Ahn, S.; Jung, S.; Lee, S. Molecules, 2013, 18, 5858-5890. 4 Chen, J.; Saeki, F.; Wiley, B.; Cang, H.; Cobb, M.; Li, Z.; Au, L.; Zhang, H.; Kimmey, M.; Li, X.; Xia, Y. Nano Lett. 2005, 5, 473-477. 5 Jamieson, T.; Bakhshi, R.; Petrova, D.; Pocock, R.; Imani, M.; Seifalian, A. Biomaterial, 2007, 28, 4717-4732. 6 Chan, W.; Maxwell, D.; Gao, X.; Bailey, R.; Han, M.; Nie, S. Biotechnology, 2002, 13, 40-46. 7 Wang, Y.; Chen, L. Nanomedicine, 2011, 7, 385-402. 8 Chithrani, D.; Ghazani, A.; Chan, W. Nano Lett. 2006, 6, 662-668. 9 Ghosh, P.; Han, G.; De, M.; Kim, C.; Rotello, V. Adv. Drug Delivery, 2008, 60, 1307-1315. 10 Pissuwan, D.; Niidome, T.; Cortie, M. Journal of Controlled Release, 2011, 149, 65-71. 11 Vasir, J.; Reddy, M.; Labhasetwar, V. Current Nanoscience, 2005, 1, 47-64. 12 Turkevich, J.; Stevenson, P.; Hillier, J. Princeton University, 1953, 57, 670-673. 13 Polte, J.; Ahner, T.; Delissen, F.; Sokolov, S.; Emmerling, F.; Thunermann, A.; Kraehnert, R. J. Am. Chem. Soc. 2010, 132, 1296-1301. 14 Haiss, W.; Thanh, N.; Aveyard, J; Fernig, D. Analytical Chemistry, 2007, 79, 4215-4221. 15 Grobenly, J.; DelRio, F.; Praeep, N.; Kim, D.; Hackley, V.; Cook, R. National Institute of Standards and Technology. 2009, 1-20. 16 He, H.; Xie, C.; Ren, J. Anal. Chem. 2008, 80, 5951-5957.